Schistosoma japonicum-induced hepatic fibrosis. A single-chain variable fragment (scFv) was generated from the murine anti-Schistosoma japonicum (S. japanicum) monoclonal antibody NP11-4. The scFv was expressed as a soluble protein and purified by Ni-affinity chromatography. After conjugation with artesunate, the binding ability with soluble egg antigens (SEA) was determined by an enzyme-linked immunosorbent assay (ELISA). The biological activity of purified scFv, scFv-artesunate (immunotoxin), and artesunate was detected in vivo. Image-Pro Plus software was used to analyze the size of egg granuloma and the extent of liver fibrosis. The recombinant scFv expession vector was constructed and expressed successfully. After purification by a His-trap Ni-affinity column, the scFv yield was approximately 0.8 mg/L of culture medium. ELISA results showed that chemical conjugation did not affect the binding activity of the immunotoxin. Our animal experiments indicated that the immunotoxin could significantly reduce the size of egg granuloma in the liver and inhibit hepatic fibrosis. The immunotoxin could be used as a promising candidate in the targeted therapy of S. japonicum-induced hepatic fibrosis.Schistosomiasis is one of the most prevalent parasitic diseases in China, and hepatic fibrosis caused by schistosome infection is the principal cause of death. The aim of this study was to evaluate the efficacy of NP11-4-derived immunotoxin scFv-artesunate on  Schistosoma, is an endemic disease in 74 developing countries. In individuals infected with S. japonicum, chronic egg-induced inflammation in the periportal tracts of the liver can lead to fibrosis, portal hypertension, bleeding, and eventually deathSchistosomiasis, which is caused by several species of the trematode S. japanicum in China. With its high efficacy and low cost, it has become the backbone of the national schistosomiasis control program in ChinaOver the past 30 years, praziquantel has been widely used for the treatment of patients infected with in vivoS.japonicum monoclonal antibody NP11-4 was prepared in our previous workH) and light (VL) chain variable regions from the NP11-4 hybridoma. The immunotoxin was constructed by chemically conjugating scFv with artesunate, and its binding activity was evaluated by enzyme-linked immunosorbent assay ELISA in vitro . The therapeutic efficacy of immunotoxin for schistosomiasis-induced liver fibrosis was determined in vivo.Immunotoxins, which couple the specificity of monoclonal antibodies (MAbs) with a highly lethal cellular toxin, have been used to selectively eliminate cell subpopulations S. japonicum, was previously established in our laboratory. The sequences of HV and LV gene were obtained in the previous work7 fresh NP11-4 hybridoma cells, using the RNA Now kit . First-strand cDNA was synthesized from total RNA using MMLV reverse transcriptase and oligo (dT) primer  according to the manufacturer's protocol. The VH regions were amplified with the primers VHF and VHR. The primers VLF and VLR were used for amplifing VL regions. The sequences of the different oligodeoxynucleotide primers are listed in H gene of anti-S. japonicum was fused to the 5′ end of the VL gene via an 18-amino acid linker sequence (GGSSRSSSSGGGGSGGGG) using the overlapping PCR method to generate the anti-S. japonicum scFv gene. The amplified scFv gene was cloned into the pMD18-T vector  and sequenced by Bioasia Co., Ltd .Hybridoma cell lines NP11-4, which produced the monoclonal antibody against NcoI and EcoRI restriction enzymes, and then inserted into the pBAD/g III A vector. E. coli Top10F′, transformed with the recombinant plasmid pBAD/gIII A-scFv was grown in a shaking culture  in a 2×YT medium supplemented with 100 µg/mL ampicillin to an OD600 of 0.8-1.0. Protein expression was induced by the addition of 0.02% L-arabinose  and 4% sucrose for 20 h at 23°C at 220 g. The cells were centrifuged at 10,000 g for 10 min at 4°C; the pellet was resuspended in phosphate buffered saline (PBS) to 1/10 of the volume of the original culture and left for 30 min on ice with sonication. After centrifugation at 12,000 g for 30 min at 4°C, the supernatant containing soluble periplasmic proteins was collected and analyzed by SDS-PAGE and Western blotting.The amplified scFv gene was digested by ™ system according to the manufacturer's protocol. The periplasmic fraction was filtered through a 0.22 µm filter, and loaded on a His-trap Ni-affinity column. Then the Ni-affinity column was equilibrated with 10 column volumes of binding buffer containing 50 mmol/L PBS, 0.5 mol/L NaCl, and 20 mmol/L imidazole. The protein of interest was eluted with the same buffer-added imidazole to 50, 100, 200, 300 and 400 mmol/L, respectively. SDS-PAGE showed that scFv was mainly eluted by a 100 mmol/L imidazole. After that, the scFv was concentrated to 1 mg/mL utilizing an amicon ultra centrifugal filter device with a 10 kD cut-off (Millipore).ScFv was purified by a His-trap Ni-affinity column and AKTApurifierTen mg artesunate was dissolved into 5 mL PBS, and 10 mg couplant A was dissolved into 1 mL dimethylformamide (DMF), and then incubated in an artesunate-couplant A mixture at 4°C for 6 h. Then 4.4 mg couplant B was dissolved in 1 mL DMF, and scFv was dissolved in 2 mL PBS. Next, The scFv-couplant B mixture was incubated at room temperature for 1 h. Subsequently, the artesunate-couplant A and scFv-couplant B were mixed 1:1 and incubated at room temperature for 1 h. The scFv-artesunate conjugation (immunotoxin) was obtained by changing the buffer system into PBS. The maximum absorption peak of immunotoxin was detected to determine whether it was conjugated.A450.For detection of the binding ability of scFv and the immunotoxin in combination with solube egg antigens (SEA), ELISA was performed by coating the 96-well plate with SEA at 10 µg/mL diluted in a sodium carbonate buffer (pH 9.6) and incubating the plate overnight at 4°C. The wells were blocked with 1% bovine serum albumin (BSA) in PBS. The primary antibodies were the purified scFv and immunotoxin diluted to 40 µg/mL with 5% non-fat milk in PBS. The secondary antibody was an anti-His antibody conjugated with peroxidase. Each step was followed by five washes with PBS plus 0.05% Tween20 (PBST). All of the immunocomplexes were revealed with 3, 3′, 5, 5′-tetramethylbenzidine (TMB) and read at S. japonicum infection was assayed by animal experiments. Eight-week-old BALB/c mice (18-22 g) were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. All mice were maintained in pathogen-free conditions and treated with standard laboratory food and water. Snails were bred and maintained in the laboratories of the Institute for Parasitic Diseases of Jiangsu Province.The therapeutic effect of the immunotoxin on the S. japonicum through abdominal skin and divided into 4 groups (15 mice/group): the immunotoxin group (immunotoxin 5 mg/kg), the scFv group (purified scFv 5 mg/kg), the artesunate group (artesunate 5 mg/kg) and the negative group (PBS). Immunotoxin, purified scFv, artesunate and PBS, respectively, were injected into the quadriceps femoris muscle on d 1, 14, and 28 after infection. Ten weeks after infection, the surviving mice were sacrificed and their livers were isolated for histological studies.Sixty male BALB/c mice were infected with 25±2 cercariae of Liver samples were fixed in 10% buffered formalin and processed for routine histopathological analysis. Four-µm sections were stained with H&E and Masson. To determine more accurately the true shape and dimension of granulomas, only those with a visible central egg were counted. A minimum of 10 granulomas were scored per liver section. The size of egg granuloma and the integrated optical density (IOD) of hepatic fibrosis were determined by computer-assisted morphometric analysis using Image-Pro Plus software .P < 0.05 was considered statistically significant.All data in the study were evaluated with the SPSS 13.0 software. One-Way ANOVA and Student-Newman-Keuls (SNK) tests were used to determine the statistical significance. A H and VL genes were amplified from the mRNA, which was isolated from the parental hybridoma cell line NP11-4. The PCR products were detected by 1% agarose gel electrophoresis  value detection was used to determine whether scFv and artesunate were conjugated. The UV absorbance of artesunate was about 210 nm, and the maximal absorption peak of scFv was 280 nm. After conjugation, the UV absorbance of the immunotoxin was about 238 nm, which was an average of the UV absorbance of scFv and artesunate. After washing by the final desalination column, the efficiency of conjugation was about 64.3%.A450 was decreased . Moreover, compared with the scFv group and the artesunate group, there was a significant difference in the immunotoxin group. However, the scFv group and the artesunate group showed no significant difference when compared with the PBS group  and variable light (VL) chains joined by a flexible linkerH and VL chains together usually vary from 10-30 amino acids in length, and the most common linker is the (Gly4Ser)3L-linker-VH orientation or VH-linker-VL orientationL primer, and a scFv was generated in VH-linker-VL form.ScFv was the smallest fragment with the original antigen-binding siteE. coli, mainly as inclusion bodies. It should be washed with a denaturation buffer, followed by denaturation and refolding in a proper buffer to harvest the active proteins. However, the refolding process does not always produce completely native protein. It is advantageous to produce proteins soluble in E. coli. The scFv we developed was expressed as soluble proteins, and the appropriate temperature for soluble periplasmic protein expression was 23°C. After purification, the yield of soluble scFv was 0.8 mg/L.The scFv was expressed as soluble periplasmic proteins and insoluble proteins in temperatures ranging from 20°C-37°C. ScFv has been produced in S. japonicum infections, a large number of eggs were incessantly produced from worms residing in the venules. The eggs elicited strong hypersensitivity reactions at the tissue sites where they were located, and the resultant destructive changes in the liver were well known as the primary etiology of schistosomiasis granuloma formationIn S.japonicum scFv and artesunate was constructed.Artesunate was considered as a vital cornerstone in the treatment and control of malaria in the pastSchistosoma agents was confirmed. The emergence of immunotoxin may solve the problem. It could target the destruction of immature and mature schistosomes and prevent their spawning, thus inhibiting granuloma formation and hepatic fibrosis. From the results of animal experiments, it appears that immunotoxin could significantly decrease granuloma size and hepatic fibrosis, compared with the other three groups. Immunotoxin may be used as a potential drug for treatment of schistosomiasis, but the specific mechanism of immunotoxin-inhibiting hepatic fibrosis requires further study.It is well known that drug resistance is unavoidable for any anti-infective compound. Recently, the existence of strains resistant to anti-S. japonicum was obtained. After purification, the yield of soluble scFv was 0.8 mg/L. The immunotoxin, including scFv and artesunate, was constructed. Moreover, the immunotoxin was applied in the treatment of schistosomiasis japonica-induced liver fibrosis, and it had sufficient therapeutic effects. The statistical results demonstrated that immunotoxin could minimize the size of egg granuloma and reduce hepatic fibrosis significantly.In conclusion, scFv of NP11-4mAb against


Background. Limiting egg consumption to avoid high cholesterolemia is recommended to reduce risk of cardiovascular disease. However, recent evidences suggest that cholesterol from diet has limited influence on serum cholesterol. Objective. To assess the effect of egg consumption on lipid profiles in hyperlipidemic adults treated with lipid-lowering drugs. Material and Method. Sixty hyperlipidemic subjects, mean age of 61 years, who had been treated with lipid-lowering drugs. Every subject was assigned to consume additional 3 eggs per day with their regular diet for 12 weeks. Measurements for lipid profiles and body compositions were performed. Results. An additional consumption of 3 eggs per day for 12 weeks increased HDL-cholesterol by 2.46 ± 6.81 mg/dL (P < 0.01) and decreased LDL-cholesterol to HDL-cholesterol ratio by 0.13 ± 0.46 (P < 0.05). No significant changes were found in other lipid profiles. Body weight and body mass index were significantly increased at 12th week by 0.52 ± 1.83 kg and 0.31 ± 0.99 kg/m2, respectively (P < 0.05). Conclusion. In hyperlipidemic adults who were treated with lipid-lowering drugs, the consumption of additional 3 eggs per day to their regular diet will increase the level of HDL-cholesterol and decrease the ratio of LDL-cholesterol to HDL-cholesterol. Cardiovascular disease (CVD) is still a major public health problem and a leading cause of death in modern world, even in Thailand . BecauseClinical trials in children, younger adults, and the elderly showed increase in both LDL-C and HDL-C with no alteration in the LDL-C to HDL-C ratio after egg consumption –9. A stuTo the best of our knowledge, no previous report on the effect of egg in hyperlipidemic adults during treatment with lipid-lowering drugs. Therefore, we try to investigate the relationship between additional 3 eggs consumption per day in these subjects with lipid profiles. Sixty subjects (58 women and 2 men) who had been diagnosed for hyperlipidemia and treated with lipid-lowering drugs with stable lipid profiles for 12 weeks were recruited. The participants who have been diagnosed as having egg allergy, hypothyroidism, coronary disease, diabetes mellitus, liver, or kidney problems were excluded from the study. An additional exclusion criterion was the regular use of hormone replacement therapy. All subjects did not change the dosage of lipid-lowering drugs during the 12 weeks of the study. All subjects were assigned to consume additional 3 eggs per day for 12 weeks and recorded amount of eggs that they consumed everyday. Subjects were not provided any other foods apart from eggs for their additional diet. Moreover energy intake was not restricted. Subjects were asked to maintain their normal routine physical activity during this study. The weight, height, body mass index, blood pressure, body composition, serum lipid profiles, 4-day dietary record, and daily egg record were performed and collected every 4-week interval. The biochemical tests including plasma glucose, renal and liver function tests, and hematological parameters were measured before and after the study.The study was reviewed and approved by the Committee on Human Research of Ramathibodi Hospital, Mahidol University. The subjects were informed in detail about the study and informed consents were obtained from all of them. Subjects completed detailed 4-day dietary records. They were required to maintain the food records for 3 weekdays and 1 weekend day. The interviewers collected the data and estimated the serving portions with the use of household measuring cups, spoons, and ruler to assist the subjects in the recall of foods eaten. During the study, the subjects were asked to check on a daily egg record sheet. The serum lipid profiles included total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) which were measured by enzymatic methods . Body composition was determined with the use of the InBody 720 body composition analyzer . Body composition was measured in the morning after an overnight fast. Body mass was recorded to the nearest 100 gram on a calibrated digital scale. Analyses were performed by the same technician who was unaware of the study details. P < 0.05 was considered statistically significant.Baseline characteristics and serum lipid profiles of all subjects were reported by using mean ± standard deviation (SD). Statistical analysis was conducted using SPSS software version 13.0 for windows. All outcome measurements among baseline and each period data were assessed using two-way repeated measures ANOVA. Sixty hyperlipidemic subjects participated in this study. Ninety-six point six percent of the subjects were female. The age of the subjects was 45 to 85 years, with a mean age of 61.8 years . Two subBody weight and body mass index were significantly increased at the 12th week. Body fat weight, lean body weight, and total body water did not show significant change as well .P < 0.01) and decreased LDL-C to HDL-C ratio by 0.13 ± 0.46 (P < 0.01). The study showed no significant change of TG (P = 0.97), TC (P = 0.62), and LDL-C (P = 0.59)  . The bioThe results of this study showed that egg consumption did not unfavorably influence serum cholesterol or other measurement of the lipid profile, in a previously noninvestigated population of hyperlipidemic adults who were treated with lipid-lowering drugs. In addition, egg consumption led to an increase in HDL-C as well as a decrease in LDL-C to HDL-C ratio, the major determinants of CVD risk. The other cohort study that has specifically examined the relationship between egg consumption and CVD included 37,851 men and 80,002 women who were free from chronic diseases of the Health Professionals Follow-Up Study and the Nurses' Health Study. After adjusting for multiple confounders, those eating >7 eggs/week (as compared with those consuming <1 egg/week) had no increased risk of CVD or stroke in either healthy men or women . In two Clinical trials conducted in healthy postmenopausal women and men aged 60 years or older have clearly demonstrated that women and men classified as hyporesponders to dietary cholesterol had no changes in LDL-C and HDL-C after consuming 3 eggs per day for 4 and 12 weeks, respectively. In contrast, those individuals who were classified as hyperresponder exhibit increases in both LDL-C and HDL-C with the result of no changes in the LDL-C to HDL-C ratio , 14. In The lipid profile after consuming additional 3 eggs per day in hyperlipidemic adults, who were treated with lipid-lowering drugs, changed similar to those of hypo-responders: no change in LDL-C, increase in HDL-C, and decrease in LDL-C to HDL-C ratio. These findings in keeping with a previous study of the effect of egg consumption in patients on HMG-CoA reductase inhibitor showed significant increases in HDL-C in subjects given 2 or 4 eggs daily . The absThe results of this study suggest that an addition of 3 eggs per day to regular diet can raise the level of HDL-C and decrease the ratio of LDL-C to HDL-C in hyperlipidemic adults who are treated with lipid-lowering drugs. The results from this study will provide added-value knowledge for simple understanding among general population about egg consumption, especially that egg cholesterol is not equal to blood cholesterol. Further testing in individuals with established CVD is now justified to clarify the place of eggs in a judicious and heart-healthy diet.


Compared to the 5-hour sleep/dim-light condition, the red, green, and blue morning light exposures significantly increased leptin concentrations . Morning red light and green light exposures significantly decreased ghrelin concentrations , but morning blue light exposures did not. This study is the first to demonstrate that morning light can modulate leptin and ghrelin concentrations, which could have an impact on reducing hunger that accompanies sleep deprivation.Acute and chronic sleep restrictions cause a reduction in leptin and an increase in ghrelin, both of which are associated with hunger. Given that light/dark patterns are closely tied to sleep/wake patterns, we compared, in a within-subjects study, the impact of morning light exposures  to dim light exposures on leptin and ghrelin concentrations after subjects experienced 5 consecutive days of both an 8-hour (baseline) and a 5-hour sleep-restricted schedule. In morning dim light, 5-hour sleep restriction significantly reduced leptin concentrations compared to the baseline, 8-hour sleep/dim-light condition ( Leptin is a hormone secreted from the white adipocytes and plays a role in the maintenance of energy homeostasis. Ghrelin is a 28-amino acid peptide hormone secreted by the stomach. Although the role of ghrelin in energy homeostasis is still unclear, its secretion depends largely on nutritional state, with ghrelin concentrations increasing prior to a meal and decreasing after eating .Both chronic and acute sleep restriction cause a reduction in serum concentrations of leptin and an increase in serum ghrelin concentrations –7. SubjeLeptin concentrations exhibit a strong circadian pattern, with the highest concentrations occurring at night and the lowest concentrations in the middle of the day. It has been shown that neither feeding time nor adrenalectomy affected the rhythmicity of leptin release. Ablation of the suprachiasmatic nuclei (SCN) in rats, where the circadian pacemaker is located, however, eliminated leptin circadian rhythmicity in rodents, suggesting that the central circadian clock regulates leptin expression . GhrelinLight has an impact on hormone production through stimulation of the SCN. Light/dark patterns, conveyed from the retina to the SCN via the retinohypothalamic (RHT) tract, are the major synchronizer of the SCN to the 24-hour solar day. The SCN controls the timing of production of a series of biomarkers, such as the timing of melatonin synthesis. Melatonin, a hormone produced at night and in darkness, is believed to be a primary timing messenger for the entire body, providing seasonal and daily photoperiod information to every neural, physiological, and cellular system. Nighttime exposures to certain light levels and spectra will cease or diminish the production of melatonin. In humans, nocturnal melatonin suppression is maximally sensitive to short-wavelength (blue) light peaking close to 460 nanometers (nm). Light can also impact hormones and brain activity without affecting nocturnal melatonin production , 11. FigFluctuations in body weight have been associated with changes in day lengths, suggesting a role of the biological clock in regulating metabolism. It has been shown that in middle-aged rats, daily melatonin administration (liquid diet containing 0.2 mcg/mL melatonin) decreased weight gain in response to a high-fat diet and decreased nighttime plasma leptin concentrations within 3 weeks of the treatment . These eThe present study was an investigation of how different light spectra might counteract the impact of sleep restriction on concentrations of leptin and ghrelin in subjects who maintained a 5-hour sleep schedule for 5 consecutive days. Since sleep restriction decreases leptin and increases ghrelin concentrations and since light has a strong influence on hormone production, such as melatonin production, it was conceivable to hypothesize that light modulate concentrations of leptin and ghrelin after sleep restriction. Specifically, as in previous studies, it was hypothesized that, after sleep restriction, morning leptin concentrations collected in dim light would be lower and ghrelin concentrations would be higher than after the baseline week (8-hour sleep opportunity). It was further hypothesized that when subjects underwent a sleep-restricted schedule (5-hour sleep opportunity per night for 5 consecutive nights), exposure to narrowband long-wavelength (red), middle-wavelength (green), and short-wavelength (blue) lights would counteract the effect of sleep deprivation on these biomarkers by increasing concentrations of plasma leptin and decreasing concentrations of plasma ghrelin. If it could be shown that narrowband lights modulate concentrations of leptin and ghrelin, a secondary goal of the study was to determine whether their spectral sensitivities were similar to that of acute melatonin suppression, which is maximally sensitive to short-wavelength light.2. All subjects included in the study were screened for medical illness and previous gastrointestinal surgery, and all reported being nonsmokers and medication-free. Shift workers or subjects who had traveled across time zones four weeks prior to the experiment were not accepted into the study. The Institutional Review Board at Rensselaer Polytechnic Institute approved the study and participants provided informed consent and were paid for their participation.Eleven subjects  participated in the experiment. The mean ± standard deviation (SD) age of participants was 27.4 ± 8.7 years and the mean ± SD body mass index (BMI) was 25.2 ± 4.2 kg/m2, peak = 633 nm, full-width-half-maximum [FWHM] = 14 nm); (2) 60 lux of green light ; (3) 60 lux of blue light ; (4) dim light conditions . Two light emitting diodes (LEDs) were mounted to each of the goggle lenses. Prior to every experiment, the light goggles were calibrated in the laboratory using a spectroradiometer  with a UV-VIS optical fiber ending in a Lambertian diffuser.Subjects experienced four morning light-exposure conditions for two hours upon wakening: (1) 60 lux of red light . Participants were required to maintain an 8-hour sleep schedule during the first baseline week. During weeks 3, 5, 7, and 9, participants were asked to maintain a 5-hour sleep schedule from Sunday through Thursday prior to data collection. During the sleep restricted weeks, subjects were asked to maintain the same wake-up times as the baseline week and they were required to go to bed 3 hours later than their bedtimes during the baseline week. Six subjects (group 1) were asked go to bed at 10:00 p.m. and wake up at 6:00 a.m. and five subjects (group 2) were asked to go to bed at 11:00 p.m. and wake up at 7:00 a.m. during the 8-hour schedule weeks. The two groups were selected based on their typical wake-up times, which were always kept the same during the experiment. During the sleep-restricted schedule weeks, the same six subjects who had a wake-up time at 6:00 a.m. were required to stay awake until 1:00 a.m. and the other five subjects who had a wake-up time at 7:00 a.m. were required to go to bed at 2:00 a.m. During weeks 1, 3, 5, 7, and 9 subjects were asked to sleep at the laboratory on Thursday evenings, and data collection occurred on Friday mornings. During weeks 2, 4, 6, and 8, participants were required to maintain a regular 8-hour sleep schedule (similar to the baseline week) so that they could return to baseline; they were not required to come to the lab at the end of those weeks.A), and circadian stimulus (CS) levels. Concisely, photopic illuminance is irradiance transformed by the photopic luminous efficiency function (Vλ), providing an orthodox measure of the spectral sensitivity of the human fovea. CLA, based on nocturnal melatonin suppression, is irradiance weighted by the spectral sensitivity of the retinal phototransduction mechanisms stimulating the SCN. CS is a transform of CLA into relative units from 0, the threshold for circadian system activation, to 0.7, response saturation, and corresponds to relative suppression of nocturnal melatonin after one hour of light exposure for a 2.3 mm diameter pupil during the midpoint of melatonin production.No naps were allowed throughout the experiment. Compliance was assured from data obtained from actigraphy and checked against their sleep log reports. Participants wore a wrist Dimesimeter during the length of the experiment. The Dimesimeter is a calibrated light meter that contains accelerometers and three sensors which record and store data in flash memory . Light dParticipants reported for the first, baseline overnight session at 9:00 p.m. (group 1) and at 10:00 p.m. (group 2) and were given an 8-hour sleep opportunity from 10:00 p.m. to 6:00 a.m. (group 1) and from 11:00 p.m. to 7:00 a.m. (group 2). During weeks 3, 5, 7, and 9, participants reported to the laboratory at 11:00 p.m. (group 1) and midnight (group 2) and were kept awake until their scheduled bedtimes, after which they were given a 5-hour sleep opportunity in complete darkness. Again, rest/activity patterns were analyzed and confirmed for compliance to the schedule. Participants were not allowed to eat or drink from 12 hours prior to awakening until the last blood draw was completed on the following morning. Caffeine and alcohol use was voluntarily restricted starting 24 hours prior to the overnight session in the laboratory.λ = 630 nm; less than 0.5 lux at the eye) for 120 minutes after morning awakening in the laboratory. For weeks 5, 7, and 9 participants were exposed to the red, green, or blue lights for 120 minutes upon awakening; light spectrum  presentation was counterbalanced across subjects.For the baseline, 8-hour sleep condition (week 1) and for the first 5-hour sleep-restricted condition (week 3), participants were in dim red light . The lower limits of detection were 0.75 ng/mL for leptin and 93 pg/mL for ghrelin. Intra- and inter-assay precisions were 5% and 7% for leptin and 6% and 16% for ghrelin, respectively. All samples from each overnight session were processed in the same batch.t-tests were performed to further investigate the significant main effects and interactions from the ANOVAs using the unnormalized data.Five (sleep-duration/light-exposure combinations) by 3 (sample times) analyses of variance (ANOVAs) were performed on both plasma leptin and ghrelin concentrations. Post hoc, two-tailed Student's t-tests using the data collected at the three sample times were used to determine whether the changes in leptin and ghrelin concentrations for each of the other four experimental conditions were significantly different than those obtained under the 5-hour sleep/dim-light condition. An a priori criterion probability for a Type I error was set at 0.05 for all post hoc statistical analyses.In addition, plasma leptin and ghrelin concentrations following the 5-hour sleep restriction in the dim-light condition were taken as the reference values for post hoc inferential statistical analyses. Plasma leptin and ghrelin concentrations at 60, 90, and 120 minutes after 8-hour sleep/dim-light condition and concentrations after light exposures  were normalized (ratio) to concentrations at 60, 90, and 120 minutes after the 5-hour sleep/dim-light condition. One-sample, two-tailed F4,40 = 3.3; P = 0.02) and of sample times . Post hoc, two-tailed Student's t-tests revealed that leptin concentrations after the morning red light exposure were significantly greater than after the 8-hour sleep/dim-light condition (P = 0.009) and after the 5-hour sleep/dim-light condition (P = 0.001). The mean ± standard error of the mean (SEM) and the median concentrations are presented in The ANOVA using leptin concentrations revealed significant main effects of sleep-duration/light-exposure combinations  and an almost significant interaction between sleep-duration/light-exposure combinations and sample times . Ghrelin concentrations increased significantly over the course of the two-hour experiment. The mean ± SEM ghrelin concentrations were 927 ± 69 pg/mL after 60 minutes, 947 ± 73 pg/mL after 90 minutes, and 961 ± 72 pg/mL after 120 minutes. Although the main effect of sleep-duration/light-exposure combinations was not statistically significant , ghrelin concentrations were the highest after the 5-hour sleep/dim-light condition and all three morning light exposure spectra reduced ghrelin concentrations. The mean ± SEM and median ghrelin concentrations are listed in The ANOVA using ghrelin concentrations revealed a significant main effect of sample times (In order to determine the changes in leptin and ghrelin concentrations by light relative to the reference condition (5-hour sleep/dim-light condition), data collected at 60, 90, and 120 minutes for the other four experimental conditions  were normalized to the data collected at 60, 90, and 120 minutes during the 5-hour sleep/dim-light condition.t-tests revealed that, with respect to the 5-hour sleep/dim-light condition, normalized leptin concentrations  were significantly higher for the 8-hour sleep/dim-light condition  and for the (sleep-restricted) red-light , green-light , and blue-light conditions . Leptin concentrations following the 8-hour sleep condition were nearly 20% higher than those following the 5-hour sleep condition, both of which were obtained under dim light. Exposure to the three morning light spectra increased leptin concentrations by more than 50% relative to the reference sleep-restricted/dim-light condition. One-sample two-tailed t-tests revealed that compared to the 5-hour sleep/dim-light condition, the normalized ghrelin concentrations  were significantly lower after exposure to morning red  and green  lights, but not following the blue-light exposure  and not after the 8-hour sleep/dim-light condition . One-sample two-tailed The present study is the first to demonstrate that narrowband, morning light exposures can modulate leptin and ghrelin concentrations, biomarkers associated with hunger, following sleep restriction. Previous studies have shown that sleep deprivation decreased overall leptin concentrations by 18% and increased ghrelin concentrations by 28% , 7. The The present results extend those in the literature by showing that light exposure in the morning can increase leptin and decrease ghrelin concentrations in sleep-restricted individuals although, again, the impact of morning light on leptin concentrations was more robust than on ghrelin concentrations. It is possible, however, that still higher light levels  are needed to reduce ghrelin concentrations. An experimental protocol similar to the one used here should be conducted to examine this possibility.It seems unlikely that the increases in leptin concentrations observed in the present study were the result of a circadian phase shift due to subjects' delayed bedtimes . Peak coThe pathways are not known through which light might modulate leptin and ghrelin concentrations. Since it has been shown that rats on a high-fat diet and treated with melatonin had a decrease in leptin concentrations compared to controls , the impAlthough we cannot determine the spectral sensitivities of light-induced leptin and ghrelin modulation in this experiment, the fact that the morning red light had a significant impact on both leptin and ghrelin concentrations strongly suggests that the long-wavelength cones contribute to these acute responses; there is no other known photoreceptor in the retina that would have sufficient sensitivity to red light to evoke the observed effects. A more extensive study should be performed to determine the spectral sensitivities of the light-induced increases in leptin and decreases in ghrelin before we can conclude that this is a cone response.Moreover, it is not known if color information relayed to the brain, rather than retinal irradiance levels, per se, is mediating these effects. Studies suggest that colors seen as “warm”  evoke feelings of arousal while colors seen as “cool”  are associated with calming feelings. The color red, for example, has been associated with feelings of danger, love, rage, and excitement as well as negative feelings such as anxiety, anger, and annoyance –22. It hThe present results need to be replicated in a larger group of subjects, but they suggest that light can have a significant impact on reducing hunger that accompanies sleep deprivation. It has been shown that obese individuals have higher leptin concentrations than lean individuals and are more resistant, or have a greater tolerance, to the effects of leptin. Animal studies should be performed to determine the pathways via which light is modulating these biomarkers and if light can change leptin resistance in obese animals. Whether light can be used as a nonpharmacological tool to clinically impact obesity should be the subject of future research.


Intriguingly, the fbaB promoter contains an imperfect PIP-box (plant-inducible promoter) (TTCGT-N9-TTCGT). The expression of fbaB was negatively regulated by a key hrp regulatory HrpG and HrpX cascade. Base substitution in the PIP-box altered the regulation of fbaB with the cascade. Furthermore, the expression of fbaB in X. oryzae pv. oryzicola RS105 strain was inducible in planta rather than in a nutrient-rich medium. Except other hrp-hrc-hpa genes, the expression of hrpG and hrpX was repressed and the transcripts of hrcC, hrpE and hpa3 were enhanced when fbaB was deleted. The mutation in hrcC, hrpE or hpa3 reduced the ability of the pathogen to acquire pyruvate and malate. In addition, bacterial virulence and growth in planta and EPS production in RΔfbaB mutant were completely restored to the wild-type level by the presence of fbaB in trans. This is the first report to demonstrate that carbohydrates, assimilated by X. oryzae pv. oryzicola, play critical roles in coordinating hrp gene expression through a yet unknown regulator.Fructose-bisphophate aldolase (FbaB), is an enzyme in glycolysis and gluconeogenesis in living organisms. The mutagenesis in a unique  For instance, the hrp expression in Ralstonia solanacearum is activated possibly by ubiquitous and non-diffusible molecules in the presence of pathogen-plant cell contact hrp system for bacterial pathogenesis in plants.Previous reports have confirmed that some carbohydrates and sulfur-containing amino acids have the ability to induce the expression of Xanthomonas species interact with plants, some of the hrp gene products generate a pedestal-like T3S structure that traverses the two bacterial membranes hrp genes is controlled by two key regulatory genes, hrpG and hrpX, which are located outside of the hrp gene cluster hrpB to hrpF promoter regions to activate transcription of hrp15–16-TTCGB- N30–32-YANNNT  in the promoter region hrpF with an imperfect PIP-box (TTCGC-N8-TTCGT) or without following the -10 box-like motif in the promoter region, have been described as being expressed in a HrpX-dependent manner Xanthomonas hrp-hrc-hpa expression is orchestrated by multiple two-component systems and transcriptional regulators such as Trh hrcC, hrpE and hpa3 genes is not obviously and completely controlled by these regulators, including HrpG and HrpX, in X. oryzae pv. oryzicola when the pathogen grows in hrp-inducing medium and in plantahrp gene expression.When X. oryzae pv. oryzicolafbaB. In this report, we present genetic evidence demonstrating that fbaB is required for gluconeogenesis, EPS production and the expression of hrp genes, as well as the full virulence of X. oryzae pv. oryzicola in rice.To investigate uncertainty above, we screened our previous Tn5-tagged mutant library of Escherichia coli strains were routinely grown in LB (Luria-Bertani) medium at 37°C X. oryzae pv. oryzicola strains were performed at 28°C in NA , NB (NA without agar), NAN (NA without sucrose) or NAS (NA with 100 g/L sucrose), NY (NB without beef extract and sucrose), the non-carbohydrate minimal medium (NCM) (2 g/L (NH4)2SO4, 4 g/L K2HPO4, 6 g/L KH2PO4, 0.2 g/L MgSO4 · 7H2O) Strains and plasmids used in this study are listed in X. oryzae pv. oryzicola and plasmids was performed as described by Turner http://www.croasia.net/company/jinsite_biotechnology_co.html). The genes cloned or amplified in this study were refered to the hrp clusters of X. oryzae pv. oryzicola RS105 strain  and the genome sequence of X. oryzae pv. oryzicola BLS256 strain (http://cmr.jcvi.org/cgi-bin/CMR/GenomePage.cgi?org=Xoc).DNA manipulation was performed following the standard procedures described by Sambrook Oryza sativa ssp. indica cv. Shanyou63, susceptible to X. oryzae pv. oryzicola RS105 strain, was used for callus induction. Seeds were dehulled and sterilized in 70% ethanol for 10 min and then in 50% commercial bleach with a few drops of Tween-20 for 30 min and then in 1% HgCl2 for 15 min. The sterilized seeds were washed 5 times with sterile distilled water and placed on N6 medium (10) with 2, 4-D (5 mg/L) for induction of rice callus at 28°C in the dark. The actively growing calli were selected and transferred to liquid N6 medium containing with 5 mg/L 2, 4-D and 1 mg/L kinetin (KT). The cells were maintained in the dark on a 7 day subculture schedule at a dilution of 1∶5 (inoculum: fresh medium). Generally, large amounts of rice suspension cells can be obtained after 4–5 week subculture and then dispersed or single round rice cells could be observed under the microscope.fabB in X. oryzae pv. oryzicola RS105 strain was constructed by using homologous recombination as described by Jiang fabB  was amplified by PCR using the total DNA of X. oryzae pv. oryzicola RS105 as the template and the primer pair fbaB-F/fbaB-R  for HR detection and into rice seedlings  for water-soaking formation with needleless syringes, respectively, and to inoculate in adult rice plants  by leaf-needling for lesion length measurement. All plants were grown and maintained in a greenhouse with 12-h day-night cycle illuminations with a fluorescent lamp and a constant temperature of 25°C with relative humidity at 75 to 80% HR and pathogenicity assays were performed as described Xanthomonas bacterial suspensions at 1×108 cfu/ml were infiltrated into the intercellular spaces of fully expanded leaves of rice  with needleless syringes at three spots on each leaf. Three 0.8 cm diameter leaf discs were harvested with a cork borer from each infiltration area after infiltration. After being sterilized in 70% ethanol and 30% hypochlorite, the leaf discs were homogenized in 1 ml of distilled water. Diluted homogenates were plated on NA agar plates supplemented with appropriate antibiotics. The number of bacterial colonies on these plates was counted after incubation at 28°C until single colonies could be counted after 3 to 4 days. The number of bacterial CFU per square centimeter of leaf area was then estimated, and the standard deviation was calculated using colony counts from the three triplicate spots from each of the three samples per time point per inoculum. Experiments were repeated at least three times.Xanthomonas bacteria were preincubated in 5 ml NB medium for 16–20 h at 28°C with shaking at 200 rpm until the OD600 value reached 0.6, and 2% of this culture was subcultured into 20 ml of the fresh NB for 16–18 h incubation. The bacterial cells were collected and washed twice, and resuspended to an optical density of 600 nm of 0.1 in 100 ml of the minimal medium NCM supplemented with different carbon source at 0.5%. For each time point, 200 ul of each culture was removed and determined by measuring OD600 against the medium blank. Data presented were from a representative experiment; the experiment was repeated independently three times.As to the detection of bacterial growth influenced by different carbohydrates, 9-TTCGT, which is not typical to TTCGC-N15-TTCGC, following 30 base pair interval space before a -10 box-like motif CAGCAT in the fbaB promoter region. Base-substitution in the PIP-box sequence of the fbaB promoter region was performed via a PCR amplification strategy. Briefly, three substituted sequences, TTCGC-N9-TTCGT (fbaBb), TTCGT-N9-TTCGC (fbaBc) and TGATA-N9-TTCGT (fbaBd) within the fbaB promoter region were generated by using three primer pairs, fbaBa-F/fbaBb-R, fbaBa-F/fbaBc-R and fbaBa-F/fbaBd-R  gene into the broad-host-range cloning vector pHM1  various sugars at 28°C with constant shaking at 200 rpm for 3 days. EPS was precipitated from the culture supernatant with ethanol, and dried to constant weight at 55°C, and weighed. Every experiment was repeated at least three times.EPS production was measured as previously described by Tang gusA, was assayed by semi-quantitative RT-PCR or real-time quantitative RT-PCR with corresponding primer pairs  according to the manufacturer's protocol. cDNA synthesis was conducted with AMV random primers (order no. D3801) provided by the manufacturer . Before synthesis of the first-strand cDNA, total RNAs were digested with RNase-free DNase I  to remove potential traces of genomic DNAs. Semi-quantitative RT-PCR was performed on the ordinary PCR apparatus and the PCR program was as follows: step 1, 95°C for 3 min; step 2, 95°C for 20 s; step 3, 55°C for 30 s; step 4, 72°C for 40 s; 35 cycles from steps 2 to 4; and step 5, 72°C for 10 min. Real-time quantitative RT-PCR was performed on the Applied Biosystems 7500 real-time PCR System using SYBR Premix Ex Taq™ , and the PCR thermal cycle condition was as following: denature at 95°C for 30 s and 41 cycles for 95°C, 5 s; 60°C, 34 s. The expression level of the 16S rRNA gene was used as an internal standard. The comparative-threshold method was used to calculate the relative mRNA level with respect to the corresponding transcript in cells cultured in NB or NY medium or rice suspension cells. All RT-PCRs were performed in triplicate.The expression of tested genes, including the reporter er pairs . XanthomfbaB gene as a virulence factor came from work aimed at the identification of genes involved in virulence alteration of X. oryzae pv. oryzicola RS105 strain in rice. The approach was to screen a Tn5-tagged mutant library of RS105 Xoryp_17640 at the 185 bp site (X. oryzae pv. oryzicola RS105 in rice (data not shown). The genome location and genetic organization of the Tn5-insertion in Xoryp_17640 of X. oryzae pv. oryzicola RS105 suggests the presence of a transcriptionally active gene Xoryp_17640 (Xanthomonas species (data not shown). It performs the reversible action of converting fructose-1,6-bisphosphate to dihydroxyacetone phosphate and glyceraldehyde 3-phosphate, which are involved in functional glycolytic and gluconeogenic pathways Xoryp_17640 is hereafter named as fbaB.The discovery of the  bp site , reducedyp_17640 . FructosfbaB, a nonpolar fbaB mutant, named RΔfbaB  symptoms, the symptoms were significantly less severe than that caused by the wild-type RS105  by approximately 1.5 cm compared to the wild-type RS105, while the T3SS mutant RΔhrcV, used as a negative control, did not cause any BLS symptoms in rice  supplemented with glucose, sucrose, fructose, mannose, galactose, pyruvate and malate, respectively, as the sole carbon source. The growth of the RΔfbaB strain grew in a similar fashion to that of the wild-type RS105 when supplemented with glucose, sucrose, mannose or galactose (data not shown). However, the growth of RΔfbaB was significantly slower than that of the wild-type strain RS105 in liquid NCM supplemented with fructose as the sole carbon source, while the complemented strain CRΔfbaB with the fbaB gene restored the growth to the wild-type level  difference in EPS production among RΔfbaB, CRΔfbaB and RS105 when they grew in NY medium alone and NY medium containing 2% of glucose, respectively  revealed an imperfect PIP-box (TTCGT-N9-TTCGT) interval by 30 bp sequence with a -10 box-like motif (CAGCAT) upstream of the fbaB start codon  in the hrpX mutant RΔhrpX and the hrpG mutant RΔhrpG than that of the wild-type RS105 when the strains grew in rice suspension cells for 16 h. The expression of fbaB in RΔhrpG was higher than that in RΔhrpX, whereas there were no obvious difference of the fbaB expression among these three tested strain when they grew in NB medium (fbaB is inducible in planta and repressed by HrpX and HrpG when the pathogen infects the host rice rather than in necrotrophic growth. The negative regulation of fbaB with HrpG and HrpX and the higher expression of fbaB in the hrpG mutant than in the hrpX mutant imply that other unkown factor(s) may involve in the regution of fbaB.Previous reports have demonstrated that the PIP-box of HrpX regulons serves as a a BLS256  by searcrt codon , suggestB medium . These rB  may increase or decrease the transcript level of a HrpX-regulated gene by up to 50% 9-TTCGT) of fbaB are affected by HrpG and HrpX, the fifth base of the first or second TTCGT motif was substituted with a C to create TTCGC by site-direct substitutions, which generated pFbaBbGUS and pFbaBcGUS  than that in RΔhrpG  affected by different carbonhydrates.The negative regulation of d malate . These rhrp regulatory genes, hrpG and hrpX, are induced or repressed when X. oryzae pv. oryzicola evidently uses sugars from plants as nutrient sources, implying that the expression of the hrp-hrc-hpa genes, which are regulated by HrpG and HrpX as reported fbaB is mutated. To investigate this, we employed a semi-quantitative . The results showed that: i) the expression of hrpG and hrpX in the fbaB mutant RΔfbaB was significantly  lower than that in the wild-type, implying that the dysfunction in glycolysis and gluconeogenesis by the mutation in fbaB represses the transcript of hrpG and hrpX; ii) the mRNA level of the tested genes, hrpD5, hrpD6, hpa1, hrpB1, hrcU and hrpF, in RΔfbaB was similar to that in the wild-type; iii) the transcriptional level of hrcC, hrpE and hpa3 , highly conserved in other Xanthomonads, and converts the intermediate fructose-1,6-bisphosphate to reversible dihydroxyacetone phosphate and glyceraldehyde 3-phosphate, which is essential for glycolysis and gluconeogenesis , but impairs the growth rate when fructose is as the sole carbon  activity essential for a functional EMP, although a putative phosphofructokinase-encoding gene is annotated in the genomes of X. oryzae pv. oryzae, X. campestris pv. vesicatoria, and X. campestris pv. campestrisfbaB of X. oryzae pv. oryzicola results in the complete loss of the capability to grow when pyruvate and malate are used as the sole carbon source . The mutagenesis in fbaB leads the complete loss of the ability to convert pyruvate and malate into glucose-1-phosphate via gluconeogenesis for EPS synthesis in X. oryzae pv. oryzicola, explaining the reason that the EPS production in the fbaB mutant is remarkably reduced when pyruvate and malate are used as the sole carbons , plant signals , and catabolite repression that may be encountered during the infection Xanthomonas-plant pathosystems and in some cases even between closely related bacteria within the same pathosystem hrpG and hrpX. Sucrose, galactose, mannose, glucose, fructose and pyruvate significantly increase the expression of hrpX in X. oryzae pv. oryzicola when they used as the sole carbon source, while sucrose and glucose remarkably enhance the expression of hrpG  suggests that the expression of fbaB may commonly be negatively regulated by HrpG and HrpX. Protein secretion assays demonstrated that FbaB is not secreted through the T2SS and T3SS (data not shown). In fact, the expression level of fbaB in the hrpG mutant is higher than than in the hrpX mutant , suggesting that HrpX, a transcriptional activator, may form a complex with a yet unknown factor to regulate the expression of fbaB. The expression of this unknown factor may be inhibited by intermediates, like malate, from the TCA cycle in X. oryzae pv. oryzicola, resulting in the lower expression of HrpG and HrpX when the block of gluconeogenesis is made by the fbaB mutation . Arrows indicate locations and orientations of the ORFs or protein IDs, and lines indicate the intergenic sequences. ▾above ORF Xoryp_17640 presents the insertion site of a transposon Tn5 derivative in mutant Mxoc0504. A non-polar construction of a fabB deletion mutant was sketched .pF/downR  and by S(TIFF)Click here for additional data file.Table S1Primers used in this study.(DOC)Click here for additional data file.


New research proposes the pluripotency of spermatogonial cells obtained from testis. These spermatogonia-derived stem cells are called embryonic stem-like cells that express embryonic stem cell markers and differentiate to the three germ layers.The aim of the present study was to generate embryonic stem-like cells from neonatal mouse testis.The Testis cells were collected from neonatal mouse. After decapsulation, testis was mechanically dissected and dissociated via a two-step mechanical and enzymatic digestion. The spermatogonia and sertoli cells were cultured together in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 15% FBS and LIF. Before one week, several small spermatogonia colonies were observed on top of the monolayer of sertoli cells. These colonies were passaged every four days. ES-Like cells colonies that resembled ES cell was appeared within 2-3 weeks (at passages 5). Real time PCR was performed to analyze the expression of a subset of pluripotency markers, as well as germ cell-specific genes. ES Like cells were confirmed with SSEA1, SOX2 and Oct4 immunofluorescence stainng as pluripotency markers.The Results showed that at fifth passages, the pluripotency genes; Nanog and c-myc have significant increase in ES-Like cells in compare with spermatogonia cells, whereas the spermatogonial markers; Stra8, mvh, and piwill2 became downregulated. In addition to these pluripotency genes, the ES cell marker SSEA-1, SOX2 and Oct4 were expressed in the ES-like cells, similar to ES cells.This researh indicates pluripotency evidence of ES-like cells derived from testis. ES-like cells shows some molecular characteristics with embryonic stem cells. Pluripotent stem cells are characterized by their extensive proliferation and self-renewal ability and they can differentiate into many different cell types, this property makes them a renewable source which are usable for the treatment of disease and developmental research , 2.DiffeSSCs have the ability of spermatogenesis which produces gamet, in addition these cells can transit to pluripotent cells which differentiate into all three germ layers . They wiThese cells can form embryonic stem-like cells and embryoid body structure in vitro that can differentiate to three germ layers , 14. ThiAccording to pervious reports showed pluripotency of testis, we designed a simple co-culture of spermatogonia with sertoli cells for derivation of ES-Like cells from neonatal mouse spermatogonia cells in vitro.Testis cells were collected from neonatal mouse (2–4 days old). After decapsulation of the testis, tissue was mechanically dissected and dissociated via a two-step mechanical and enzymatic digestion with DMEM medium which contained; 0.5 mg/ml collagenase-dispase, 0.5 mg/ml trypsin and 0.05 mg/ml DNase, (with shaking and pipetting) at 37°C for 30-45 min.The obtained cells that included spermatogonia and sertoli cells were incubated together at 37°C and 5% CO2, in a humidified atmosphere, they were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen) supplemented with 15% FBS, 1 mMLglutamine (Invitrogen), 0.1 mM nonessential amino acids (Invitrogen), 0.1 mM-mercaptoethanol and LIF. Two days postplating, most testicular cells were attached to the growing surface, and medium was changed. Before one week, several small spermatogonia colonies were observed on top of the monolayer of sertoli cells. These colonies were passaged every four days. Finally, ES-Like cells colonies that resembled ES cell was appeared within 2-3 weeks (at passages 5). The number of the cells was determined using a hematocytometer before culture. Cell viability was evaluated by means of dye exclusion test (trypan blue solution). ES-Like cells were confirmed with Real Time- PCR and Immunoflurcent staining. Embryonic stem cells considered as a control group.EBs was created using hanging drop method. ES-Like cells were lifted, washed, and resuspended in LIF-free DMEM supplemented with 5% FBS to a concentration of 2000 cells per 20μl. Twenty micro liter drops of the suspension were placed on the lid of a 10-cm plastic culture dish . The lid was turned upside down and placed on the bottom part of dish, which was filled with sterile water, creating hanging drops. The dishes were incubated at 37°C in 5% CO2. EBs were cultured for 48 h.For immunofluorescence, the cells were fixed in 4% paraformaldehyde (PFA) in PBS (pH 7.4) for 20 minutes. The cells were washed twice with PBS/0.1% Tween-20 to remove residual fixative and incubated in 1%Triton X in PBS for 30 minutes, prior to blocking in 4% normal goatserum in PBS for 30 minutes followed by incubation with antibody solution overnight at 4°C. Primary antibodies included: stage-specific embryonic antigen 1 (SSEA1) , SOX2  and Oct4 . In the following day, the cells were washed twice with PBS, and incubated with appropriate secondary antibody  in PBS. After two washes with PBS for 5 minutes, the cells were mounted with anti-fade mounting 4, 6-diamidino-2-phenylindole (DAPI)/PBS and viewed on a Leica microscope.Real time PCR was performed to analyze the expression of a subset of pluripotency markers, as well as germ cell-specific genes. Nanog and c-myc were evaluated as of pluripotency genes in ES-Like cells and the spermatogonial markers Stra8, mvh and Piwill2 were evaluated.On the basis of our experience with isolating SCs from neonatal mice, we were able to identify the cells morphologically. Two days postplating, most testicular cells were attached to the growing surface. Approximately before 1 week, several small colonies were observed on top of the monolayer of testicular cells . These cFor a more detailed examination of ES-like colonies, we performed immunocytochemical analysis and compared them with spermatogonial cells. ES-like cells expressed the cell-surface marker SSEA-1 (stage-specific embryonic antigen-1), SOX2 and Oct 4 that characterized undifferentiated mouse ESCs .Real time PCR was performed to analyze the expression of a subset of pluripotency markers, as well as germ-cell specific genes, in the isolated ES-Like cells. The results demonstrated that at fifth passages, in compare with spermatogonia cells, the pluripotency genes Nanog and C-myc has significant increase in ES-Like cells. Also the expression of Nanog and c-myc of these cells are similar to ES cells. Gene expression levels before and after the transition of cultured SCs to ES-like cells shows that the expression of spermatogonial markers Stra8, mvh, and piwill2 were down regulated, but they couldn’t reach to ES level genes expression , 4 and 5The ability of EB formation in vitro is one of the characteristics of pluripotent ES-like cells. EB formation was detected after 2–4 days of hanging drop method .Generation of ES-like cells from testis by standard methods has many application in formation of cell lines from specific patient or disease that can be used in basic research and allowing sufficient cells for regenerative therapy . In addiThe perspective of the results obtained in mice has clinical application in other species particularly in humans. There are different methods to induce ES-like cells; Kanatsu-Shinohara and Seandel used the same culture protocol, with some differences in feeder layers and time duration of emergence of ES cell-like cells , 19. GuaTime duration of emergence of ES-like colonies depends on the used culture system. Seandel used culture system with testicular peritubular and sertoli cells as a feeder layer. Guan method is the longest time in which no feeder layer was provided , 19.In cThese ES-like cells have the same molecular profiling with embryonic stem cells , 23. Our


Mycobacterium avium, host defence may depend on intracellular iron sequestration mechanisms. Ferritin, the major intracellular iron storage protein, plays a critical role in this process. In the current study, we studied ferritin expression in mouse bone marrow-derived macrophages upon infection with M. avium. We found that H-ferritin is selectively increased in infected macrophages, through an up-regulation of gene transcription. This increase was mediated by the engagement of Toll like receptor-2, and was independent of TNF-alpha or nitric oxide production. The formation of H-rich ferritin proteins and the consequent iron sequestration may be an important part of the panoply of antimicrobial mechanisms of macrophages.Important for both host and pathogen survivals, iron is a key factor in determining the outcome of an infectious process. Iron with-holding, including sequestration inside tissue macrophages, is considered an important strategy to fight infection. However, for intra-macrophagic pathogens, such as  IRE-IRP complexes were resolved in 6% nondenaturing polyacrylamide gel. Samples were treated in parallel with 2% 2-mercaptoethanol prior to the addition of the 33P-labeled IRE probe to fully activate IRP-IRE binding activity. BMM were washed with cold PBS and incubated for 10 min on ice with digitonin (Sigma) at 0.007% in sucrose 0.25M and Hepes 0.1M, pH 7.4, and centrifuged 10 min at 1800 g. The supernatant was centrifuged for 1h at 100 000 g and the mitochondria-free cytosolic extract was collected. The samples were kept at -80 °C until use. RNA-protein interactions were performed as described previously ,16 usingData was analysed using a two-tailed unpaired student’s t test. M. avium infection alters intracellular ferritin levels, we infected mouse bone marrow-derived macrophages (BMM) and analysed the ferritin protein levels for up to 5 days. The infection led to a 5.3±0.9 fold increase of ferritin heavy chain in the first 24h, which remained elevated until day 5 (3.8±0.4 fold increase) (To test whether ncrease) . As for ncrease) . M. avium were pulsed with 55Fe ferric ammonium citrate and iron incorporation in ferritin was analysed in a native PAGE followed by exposure to autoradiography film. As observed in 55Fe.We then tested if the increase in ferritin induced by infection leads to an increased diversion of iron into storage. BMM infected or not with Fth1, coding for H-ferritin, was found to be up-regulated by 5.2±1.9 fold in infected BMM at 24h (Ftl1), in accordance with the protein, was less regulated, with a reduction of 65% at day 2 of infection . The binding of IRPs to the unique iron-responsive elements (IRE), which is present in the 5’ untranslated region of both L- and H-ferritin mRNAs, blocks the mRNA translation . TherefoM at 24h , correlaM at 24h . The gennfection .Fth1 transcription [M. avium infection is a consequence of TNF-alpha production, we infected BMM genetically deficient in the production of TNF-alpha and followed the ferritin expression. As can be seen in The infection of BMM with mycobacteria induces TNF-alpha production , a cytokcription . To inve-/- BMM exhibited similar ferritin protein profile changes to those of WT BMM post infection, demonstrating that NO is not involved in the regulation of ferritin by M. avium.The infection of BMM with various mycobacteria also induces nitric oxide (NO) production . Moreove-/- macrophages and measured the cell ferritin content during the infection. Although TLR-2-/- BMM increased H-ferritin in response to infection, this effect was markedly reduced in comparison with WT BMM. The absence of TLR-2 reduced by 62% the increase of H-ferritin after 24h of infection -2 is the main receptor for the recognition of several mycobacterial constituents . To evalnfection  and as mnfection . In acconfection . Further than macrophages infected with non pathogenic species [-/- BMM infected with M. avium similarly to that of WT infected BMM. Our results further show that activation of TLR-2 increases H-ferritin through the activation of gene transcription, independently of NO. A recent report demonstrated that LPS (a TLR4 ligand) in combination with IFN-gamma strongly induced H-ferritin in IRP1-/- BMM via endogenous NO production while H-ferritin content was maintained low in WT BMM [Our results, obtained with primary mouse macrophages, showed that the infection with  species . Accordin WT BMM . TherefoM. tuberculosis by NO-dependent mechanisms [M. avium [-/- BMM were still able to increase H-ferritin in response to M. avium infection, albeit to a much lesser extent. This suggests that other Pattern Recognition Receptors could have a minor role in the recognition of M. avium and consequent induction of H-ferritin.TLR-2 is the main receptor for the innate recognition of several mycobacterial constituents . TLR-2 achanisms  and in bM. avium  by mechaM. avium infection had a much less pronounced effect on the levels of L-ferritin, overall resulting in the formation of ferritin proteins richer in H-chain. This may have important physiological consequences, since a higher H/L ratio has been shown to reduce the cell’s labile iron pool [M. avium-infected macrophages have a higher capacity for the incorporation of iron into cytosolic ferritin. Our results suggest, therefore, that H-ferritin up-regulation during infection may contribute to limit iron availability to mycobacteria, taking part of the macrophage nutriprive mechanisms [M. avium  . Severalhepcidin . M. avium by macrophage TLR-2 leads to the increase of the expression of H-ferritin, through transcriptional activation, by mechanisms that although not completely elucidated, are independent of TNF-alpha and NO. TLR2 mediated-up regulation of H-Ft in primary macrophages may be important in host protection against mycobacterial infections, by causing pathogen iron starvation and host cell protection and may also contribute to the development of ACD during mycobacterial infections. These findings can help better understand the tight regulation of host iron metabolism which occurs during the innate immune response to infections.In summary, this work shows that the recognition of Figure S1Mycobacterium avium infection on the oxidation of peroxiredoxins (Prx).Effect of  Bone marrow-derived macrophages were obtained from C57Bl/6 mice and infected with M. avium, as described in Material and Methods, or left uninfected. To evaluate oxidative damage, macrophages were lysed after 1 or 3 days and PrxSO3, formed by the overoxidation of Prx, was detected by western blot. Uninfected macrophages exposed to 100 µM H2O2 for 20 min were used as a positive control.(DOCX)Click here for additional data file.Table S1Mycobacterium avium infection on intramacrophagic ferritin.Effect of  Bone marrow-derived macrophages were obtained from C57Bl/6 mice and infected with M. avium or left uninfected. At different time points macrophages were lysed and the amount of ferritin was quantified by ELISA. Day 0 refers to the time point immediately after infection. Data are presented as ng of ferritin per mg of total protein. The results are shown as average ± SD from one experiment performed in triplicate out of four independent experiments.(DOCX)Click here for additional data file.


Here we present bioinformatic analysis on putative HQ-forming sequences (PHQS) in the genomes of eukaryotic organisms. Starting from amphibian, PHQS motifs are concentrated in the immediate 1000-nt region downstream of transcription start sites, implying their potential role in transcription regulation. Moreover, their occurrence shows a strong bias toward the non-template versus the template strand. PHQS has become constitutional in genes in warm-blooded animals, and the magnitude of the strand bias correlates with the ability of PHQS to form HQ, suggesting a selection based on HQ formation. This strand bias is reversed in lower species, implying that the selection of PHQS/HQ depended on the living temperature of the organisms. In comparison with the putative intramolecular G-quadruplex-forming sequences (PQS), PHQS motifs are far more prevalent and abundant in the transcribed regions, making them the dominant candidates in the formation of G-quadruplexes in transcription. Collectively, these results suggest that the HQ structures are evolutionally selected to function in transcription and other transcription-mediated processes that involve guanine-rich non-template strand.Recently, we reported the co-transcriptional formation of DNA:RNA hybrid G-quadruplex (HQ) structure by the non-template DNA strand and nascent RNA transcript, which in turn modulates transcription under both  G-quadruplex, a four-stranded secondary structure formed by guanine-rich (G-rich) nucleic acids, is gaining increasing attention owing to its potential role in physiological and pathological processes . DNA G-qin vitro and in vivo conditions. We further showed that putative HQ-forming sequences (PHQS) are present in >97% of human genes and their number correlate with the transcriptomal profiles in human tissues  linked by three loops A. On the tissues . These rTo further explore the physiological implication and characterize the occurrence of PHQS motifs in genomes, we carried out genome-wide analysis to organisms whose genomic data are currently available in the Ensembl genes database. Here we show that PHQS is present in much greater prevalence and abundance than the PQS. Like the PQS, PHQS motifs are also concentrated near TSS. HQ formation requires G-tracts from the non-template strand. In accordance with this, PHQS motifs exhibited preferential enrichment on the non-template strand. Our data suggest that this strand bias might be selected by a mechanism based on the capability of PHQS to form HQ. Analysis across different organisms illustrates that a negative selection of PHQS occurred in the genomes of metazoa and pisces. In contrast, a positive selection began to merge in amphibians and PHQS became constitutional in genes in warm-blooded animals. Collectively, these results suggest that HQ structures are evolutionally selected to function in transcription regulation and other transcription-mediated processes that involve the transcription of DNA with guanine-rich non-template strand, such as immunoglobulin class switching, recombination, genomic instability and replication initiation.Mustela putorius furo, which was from release 69) via the BioMart (version 0.7) interface (http://www.ensembl.org/) by selecting protein-coding in the Gene type filter and Unspliced (Gene) in the Attribute/Sequences panel. Only unique results were downloaded.Sequences of protein-coding genes and their upstream flanking region were downloaded in fasta format, respectively, along with their IDs from the Ensembl genes database  developed using the Active Perl 5.14.2 (downloaded from www.activestate.com/activeperl) under the Windows OS. The program used a pattern-matching code G{3,}{1,} to detect the sequences G≥3-(N1–7-G≥3)≥1, where G denoted guanine and N denoted any nucleotide, including G. The use of non-greedy quantifiers for loops while the rest operators were greedy by default ensured that G-tracts would not be ignored or treated as loop. Putative G-quadruplex sequences (PQS) were identified in the same way, but using the pattern-matching code G{3,}{3,} that detects sequences G≥3-(N1–7-G≥3)≥3. Each match returned the matched sequence, its coordinate . The files can be opened in Excel or similar software for viewing and further processing. Isolated G3 tracts were analyzed similarly using the pattern-matching code G{2,}{0,} that identifies all motifs with one or more G-tracts of two or more consecutive guanines, connected by loops of 1–7 nucleotides. Any found motifs that had more than one G-tract, or G-tract with size ≠ 3 were discarded. The remaining motifs are single G3 tracts isolated from other G-tracts by more than seven nucleotides. They were then processed in the same way as the PHQS.PHQS was identified with a home-made Perl program (http://genome.ucsc.edu/) from the assembly hg19, track CpG islands, in BED format. Three BED files for the G-rich TFBS, EGR1, MAZ and SP1, was downloaded using the UCSC Table Browser from the assembly hg19, track Uniform TFBS, respectively. The gene list was imported into the MotifLab to download the correspondent DNA sequences and the BED file for the motifs was imported to mask the sequences. Masked sequences were then analyzed by the homemade Perl script . Masking of heterogeneous nuclear ribonucleoproteins (hnRNP) A and H motifs was conducted using a home-made Perl script that searched for the motifs in the DNA sequences and converted them to N's as described (Motif masking for CpG islands and G-rich transcription factor binding sites (TFBSs) was conducted using the software MotifLab version 1.07 . A list escribed .2, 10 mM dithiothreitol (DTT), 2 mM spermidine, 2 mM nucleoside triphosphate (NTP) and 0.5 U/µl pyrophosphatase, inorganic . After transcription, the sample was diluted with equal volume of stop solution containing 40% (w/v) PEG 200, 50 mM KCl, 1 µM competitive DNA , 0.8 µg/µl RNase A and 0.4 U/µl RNase H, followed by a incubation at 37°C for 1 h. The reaction was terminated by addition of 1/25 vol of 0.5 M EDTA and 1/20 vol of 2% sodium dodecyl sulfate. The DNA was then resolved on a 10% polyacrylamide gel containing 75 mM KCl and 40% (w/v) PEG 200, at 4°C, 8 V/cm, in 1× tris-borate-EDTA (TBE) buffer containing 75 mM KCl. Resolved DNA was detected by the fluorescence of carboxyfluorescein (FAM) dye labeled at the 5′ end of the non-template strand using a Typhoon 9400 phosphor imager .dsDNA carrying a T7 promoter and an indicated downstream G-core was prepared by overlap extension polymerase chain reaction. Transcription was carried out essentially as previously described  at 37°C ≥3-(N1–7-G≥3)≥1; that is, two or more G-tracts of three or more consecutive guanines, connected by loops of 1–7 nucleotides. Because the formation of HQ in transcription requires a minimum of two G-tracts from the DNA strand, our searching pattern was adopted from the one G≥3 -(N1-7-G≥3)≥3 that has been used in searches for PQS in genomes in the original (≥3 -(N1-7-G≥3)≥1 and can be clarified into one or more of the other three categories. For example, a G15 can be regarded either as a 2G sequence of G7-L1-G7 or as a 4G sequence of G3-L1-G3-L1-G3-L1-G3 or others, where L1 designates a 1-nt (G) loop. Because of their small amount (<1% of PHQS in human genes) and multiple clarifications, they were not used for further frequency analysis. To survey the occurrence of PHQS in genomes, we carried out computational searches in the protein-coding genes in species in the Ensembl genes database. The search algorithm found all motifs that match the sequence pattern Goriginal ,7 and maoriginal ,34–37 bySimilar to the PQS motifs ,23,26,38Supplementary Figure S2). However, such a strand bias was not obvious for the species in the pisces and metazoan categories. More precisely, a reversed negative selection could be noticed in most of these species in which the occurrence of PHQS was higher in the template than in the non-template strand. One exception was the Latimeria chalumnae in the pisces, which also displayed a higher occurrence in the non-template than in the template strand, like the two of the amphibian species. The selection in those three species was disturbed by random noise in the background. This might reflect an evolutional transition from lower to higher organisms in the selection for PHQS.If the strand bias for PHQS is selected for a biological function, it should be conserved across related species. To trace its evolutional selection, we searched for PHQS in the genomes of all the species currently available in the Ensembl database. It can be seen that the species in the mammalian, avian, reptilian and amphibian categories showed biased positive selection for PHQS on the non-template strand, downstream of TSS for the 2G, 3G  and 4G+ Overall, an enrichment of PHQS near TSS and strand bias toward PHQS on the non-template strand began to show up in amphibians, and they became a general feature in the warm-blooded animals . In contrast, lower organisms showed much lower occurrences of PHQS than higher organisms. The reservation of a strand-biased enrichment of PHQS across the warm-blooded species argues that PHQS motifs are evolutionally selected.Promoters overlap TSS  and ofteUnlike the above elements, the G-rich motifs in RNA transcripts recognized by hnRNP A and hnRNP H is non-template specific. Therefore, the masking in this case was conducted only for the G-rich elements. This manipulation reversed the strand bias in the entire region for human (3 under any condition (lanes 1–4). For the DNAs containing two or three G3, however, HQ was observed when the transcription was carried out with normal GTP . In contrast, no HQ was seen when the DNA was subjected to a heat denaturation/renaturation  or transcribed with the GTP being substituted by 7-deaza-GTP (dzGTP) , a GTP analog used to prevent RNA from participating in G-quadruplex formation (3 tracts formed more HQ (60%) than the DNA with two G3 tracts (20%). When the number of G3 tracts increased to four, more G-quadruplex formed . Because this DNA alone was also able to form intramolecular G-quadruplex , it is not known how much HQ formed in this DNA. The G-quadruplex structures detected were those that remained after the posttranscription processing and might not exactly reflect their amount formed during transcription. However, the higher HQ amount detected in the DNA with three G3 tracts than in that with two G3 tracts implies that more G-tracts led to more chance of HQ formation, which provides an intuitive explanation to a greater PHQS strand bias for the 3G than for the 2G motifs , resulting in negative strand biases (Several lines of evidences  and 6 imd biases . The conCaenorhabditis elegans, PHQS is only present in 33% of the genes, with an average of 0.6 PHQS per gene, in sharp contrast to the human genome. In Saccharomyces cerevisiae, PHQS is only found in 27.4% of the genes, with an average of 0.36 PHQS per gene. The large difference between the lower and higher organisms in the number of PHQS motifs per gene implied that the HQ might modulate transcription through different mechanisms. In the lower organisms, HQ may serve as recognition element, as intramolecular G-quadruplex does, that functions through binding with regulatory proteins (in vitro and in vivo conditions, and the occurrence frequency of PHQS motifs in genes correlates with the transcriptional profiles in human tissues. As assumed in our previous work, HQ may regulate transcription in an intrinsic, direct and cost-effective way. We hypothesized that HQ structures may provide a general primary cis control at the root level of transcription to limit the expression potential of the host genes (HQ structures may function in two aspects. In general, lower organisms have fewer PHQS motifs per gene than higher organisms. In proteins . This fust genes .We expect that HQ should also have functionality in other processes that involve transcription. Strand-biased enrichment of guanine residues is featured in many physiologically important genomic elements, including immunoglobulin class switching sequences , prokaryThe requirement of a minimum of two G-tracts instead of four allows PHQS motifs to occur at a much higher frequency than the PQS; thus, they are the dominant candidates for G-quadruplex formation in transcription in cells . The preSupplementary Data are available at NAR Online.Ministry of Science and Technology of China ; National Science Foundation of China [30970617 and 21072189]. Funding for open access charge: Ministry of Science and Technology of China [2012CB720601].Conflict of interest statement. None declared.


Plasmodium falciparum, which is the most virulent malaria parasite infecting human being. By using Illumina/Solexa paired-end sequencing of an is-ncRNA-specific library, we performed a systematic identification of novel is-ncRNAs in intraerythrocytic P. falciparum, strain 3D7. A total of 1,198 novel is-ncRNA candidates, including antisense, intergenic, and intronic is-ncRNAs, were identified. Bioinformatics analyses showed that the intergenic is-ncRNAs were the least conserved among different Plasmodium species, and antisense is-ncRNAs were more conserved than their sense counterparts. Twenty-two novel snoRNAs were identified, and eight potential novel classes of P. falciparum is-ncRNAs were revealed by clustering analysis. The expression of randomly selected novel is-ncRNAs was confirmed by RT-PCR and northern blotting assays. An obvious different expressional profile of the novel is-ncRNA between the early and late intraerythrocytic developmental stages of the parasite was observed. The expression levels of the antisense RNAs correlated with those of their cis-encoded sense RNA counterparts, suggesting that these is-ncRNAs are involved in the regulation of gene expression of the parasite. In conclusion, we accomplished a deep profiling analysis of novel is-ncRNAs in P. falciparum, analysed the conservation and structural features of these novel is-ncRNAs, and revealed their differential expression patterns during the development of the parasite. These findings provide important information for further functional characterisation of novel is-ncRNAs during the development of P. falciparum.Intermediate-size noncoding RNAs (is-ncRNAs) have been shown to play important regulatory roles in the development of several eukaryotic organisms. However, they have not been thoroughly explored in  Plasmodium species and is as high as 98.5% in humans etc.C. elegans, they were found to be expressed in a highly stage-specific manner Bombyx mori), chicken  and rhesus monkey (Macaca mulatta), is-ncRNAs appear to play significant roles in lineage/species specification It is evident that the vast majority of the DNA in eukaryotic genomes is transcribed into noncoding RNAs (ncRNAs) Plasmodium falciparum  is the most lethal causative agent of human malaria P. falciparum 3D7 was sequenced in 2002 T. gondii and G. intestinalisP. falciparum by traditional cloning and sequencing methods P. falciparum genome P. falciparum. In 2009, Raabe et al. screened the P. falciparum genome for ncRNAs ranging in size from 10–500 nt using a cloning method and identified 298 is-ncRNAs among 630 ncRNAs from 25,000 cDNA clones et al. sought to investigate long noncoding RNAs longer than 200 nt in P. falciparum via a tiling microarray P. falciparum DNA might be lost during the cloning process, and the 14 chromosomes of P. falciparum might not be fully covered by the probes in the tiling array study, we try to explore the whole profile of is-ncRNA in P. falciparum by using the RNA sequencing. We constructed an is-ncRNA specific library of intraerythrocytic P. falciparum 3D7 with rRNAs and U1–U6 RNAs depleted, and sequenced the library using Illumina/Solexa paired-end sequencing. After obtaining 1,198 novel is-ncRNA candidates, we analysed them for conservation and secondary structure classifications. Then, we used RT-PCR and northern blotting assays to confirm the expression of the molecules. The stage-specific expression patterns of a few randomly selected is-ncRNA candidates were detected by qRT-PCR. Our data further expands the understanding of the P. falciparum is-ncRNAs that are expressed during the erythrocyte stage, reveals the expression profiles of these ncRNAs, and suggests potential roles of these molecules in regulating the development of malaria parasites.Malaria remains one of most severe infectious diseases worldwide, infecting an estimated 207 million people and causing 627,000 deaths annually . The unicellular eukaryotic parasite P. falciparum 3D7  and subjected to Illumina/Solexa paired-end sequencing  using Bowtie P. falciparum to date . Among the novel is-ncRNAs, the anti_ncRNA is-ncRNAs were the most conserved and 87.5% of them had average phastCons scores>0.5. The intergenic and intronic is-ncRNAs were much less conserved (P<0.01) than the RNAs from other loci, and nearly 70% of the intergenic is-ncRNAs had average phastCons scores≤0.2   . These fTo test the expression of the novel is-ncRNAs, 78 randomly selected novel is-ncRNA candidates were subjected to reverse transcription polymerase chain reaction (RT-PCR). Sixty of these tested novel is-ncRNA candidates , accountcis-encoded sense RNAs  in both the early (ring stage) and late stages (late trophozoites and schizonts). The analysis included nine intergenic is-ncRNAs and four antisense is-ncRNAs. Eleven of the thirteen is-ncRNAs showed significantly higher expression levels in the early stage than in the late stage; the remaining two intergenic is-ncRNAs, nc-669 and nc-716, were highly expressed in the late stage . To confnse RNAs  during tnse RNAs , consistP. falciparum is-ncRNA candidates were identified using a novel approach. We constructed an is-ncRNA library that was specifically depleted of rRNAs and U1–U6 RNAs and then sequenced this library using paired-end Illumina/Solexa paired-end sequencing. A total of 1,757 P. falciparum is-ncRNAs have been identified in this work and previous studies, greatly expanding our knowledge of the intermediate-size transcriptome of P. falciparum. These novel is-ncRNAs could not have been successfully identified without this strategy. The depletion of highly abundant rRNAs and U1–U6 RNAs during the construction of the is-ncRNA library allowed the efficient detection of novel is-ncRNAs within the intermediate-size RNA sample, as suggested by the fact that our data overlapped well (80.9%) with the is-ncRNAs  found in PlasmoDB 10.0. Furthermore, the high-throughput sequencing technique has revolutionised the field of transcriptomics by facilitating RNA analysis through cDNA sequencing at unprecedented depths and massive scale compared to traditional cloning methods Plasmodium genome C. elegans genome P. falciparum genome In this study, a total of 1,198 novel The poor overlap with is-ncRNAs identified in previous studies may have occurred for one or more of the following reasons: (1) the size-fractionated RNA sample in the study that utilised the cloning method was treated with tobacco acid pyrophosphatase P. falciparum is-ncRNA candidates, as reflected by their phastCons scores, was dependent on the genomic locations of the molecules. For instance, both the anti_mRNA exon is-ncRNAs and anti_ncRNA is-ncRNAs were shown to have a high degree of conservation, indicating the important role of these antisense RNAs during evolution, for functional sequences tend to be actively conserved throughout evolution Plasmodium species, which likely reflects the divergence within the genus PlasmodiumPlasmodium species exhibits a restricted host range  P. falciparum may be divided into eight classes, providing a framework with which to predict the targets of these is-ncRNAs. Additionally, two internal motifs, IM1 and IM2 identified in this study may facilitate further investigation of the molecular interaction between these novel is-ncRNAs and transcription factors. For example, novel is-ncRNAs with IM1 may be involved in the regulation of parasitic gene expression by interacting with serine/arginine-rich splicing factor 1, requiring further experiments to investigate.In our work, the conservation of the novel P. falciparum, suggesting that the expression of the is-ncRNAs themselves is tightly regulated. The phenomenon that eleven out of the 13 randomly selected is-ncRNAs were more highly expressed in the early stage than in the late stage is quite interesting, since parasites undergo a fast development and remarkable differentiation within less than two days cis-encoded sense RNAs were shown to be positively co-regulated during the intraerythrocytic stage. Natural antisense transcripts (NATs) have been considered to negatively regulate the expression of their cis-encoded mRNA counterparts in both P. falciparumToxoplasma gondii because a significant inverse correlation has been observed between the sense and antisense tag counts from single loci using SAGE technology cis-encoded sense RNAs in P. falciparum were reported by Raabe et al. and Sims et al.P. falciparum.In this study, significant differences in expression patterns were observed between the early and late stages of all nine intergenic and four antisense novel is-ncRNAs in PlasmodiumP. falciparum and this regulation may be unique to malaria parasites.Because the conventional RNAi pathway has not yet been identified in the P. falciparum known is-ncRNAs and provides the first evidence that they may be classified into eight groups according to their secondary structures. The characterisation of the phase-specific expression of these is-ncRNAs implies that they may play critical roles in regulating gene expression during the development of the intraerythrocytic malaria parasite. Our study also highlights candidate avenues for future functional investigations, such as is-ncRNA overexpression or knockdown experiments, the validation of the interaction between is-ncRNAs and transcription factors and ubiquitin-protein ligase, and the mechanism by which antisense is-ncRNAs regulate and control parasite gene expression. A clear understanding of these topics will provide insights that may contribute to the eradication of this parasite.Our work significantly increases the number of P. falciparum strain 3D7 was cultured in complete medium using the standard growth conditions originally described by Trager and Jensen To isolate the parasites, harvested cultures were treated with 0.1% saponin, and the released parasites were pelleted by centrifugation and washed twice with ice-cold phosphate-buffered saline (PBS). Total RNA was extracted using the TRIzol Reagent (Invitrogen) according to the manufacturer's instructions. The RNA was quantified with a NanoDrop 1000 Spectrophotometer (Thermo Scientific), and the RNA quality was confirmed using 6% denaturing urea polyacrylamide gel electrophoresis (PAGE).UCGGAAGAGCGGUUCAGCAGGAAUGCCGAG-3GA) and reverse transcribed with a universal primer . The 200–500 nt fraction were directly reverse transcribed with a random primer (5-CTCGGCTTTCCTGCTGTTCCGNNNNNN-3). Another adaptor (5-ACACTCTTTCCCTACACGACGCTCTTCCGATCT-3) was ligated to the 3′ends of the two cDNA samples. PCR was performed, and the amplified products were sequenced on a standard Illumina/Solexa paired-end sequencing platform.The is-ncRNA-specific library was constructed as previously described http://plasmodb.org/common/downloads/release-10.0/Pfalciparum3D7/) using Bowtie http://areslab.ucsc.edu/data_downloads/) after sequences of the contigs were mapped to the UCSC Malaria Genome (2007). Additionally, ncRNAs that had been reported or analysed in five previous studies http://hmmer.janelia.org/) server. A structure-based analysis of the is-ncRNAs was performed using LocARNA-based clustering The 5′ and 3′ adaptors in the raw paired-end sequencing data were removed, and reads with abnormal adaptors or inserted sequences shorter than 30 nt were filtered. For all the remaining clean reads, the first 51 bp of each read were retained and were mapped to PlasmoDB 10.0 (RT-PCR was performed using the RevertAid™ First-Strand cDNA Synthesis Kit (Fermentas) according to the manufacturer's recommendations. Specific primers see  were desRNA probes were labelled and transcribed in vitro with SP6 RNA polymerase (Promega) and dig-11-UTP (Roche). Total RNA samples (10 µg) were resolved by 7M urea 6% denaturing PAGE gel electrophoresis and transferred onto a nylon membrane  by capillary transfer in 20×SSC for at least 6 hours. The RNA sample on the membrane was then fixed by UV crosslinking. Next, the membrane was rinsed briefly in double distilled water. The membranes were subsequently hybridised with the DIG-labelled RNA probes in Hyb buffer (Roche) at 55°C overnight. Northern blotting was performed according to the manufacturer's instructions. After hybridisation, the blots were treated with Washing and Blocking Buffer (Roche), immunologically detected with anti-digoxigenin-AP, and equilibrated in detection buffer. Finally, the blots were incubated with the chemiluminescent substrate CDP-star (Roche) and exposed to Kodak film. A DIG-labelled U5 RNA probe was used as an internal control. The primers used for DIG-labelled RNA probe transcription are listed in P. falciparum 3D7 was reverse transcribed. qRT-PCR was performed with 1∶10 diluted cDNAs, a StepOnePlus™ Real-Time PCR System (Applied Biosystems) and TransStart Green qPCR SuperMix (Transgen) under the following cycling conditions: 95°C for 5 min; 40 cycles of 95°C for 15 s, 55°C for 20 s and 72°C for 20 s; and a final extension at 72°C for 5 min. A melting curve was generated as follows: 95°C for 15 s, 55°C for 1 min followed by a temperature gradient with a ramp rate of 0.5°C/s, and a final denaturation at 95°C for 15 s. The obtained products were also evaluated on a 6% denaturing urea PAGE gel. For relative quantification, a house-keeping gene  was used as an internal control. For the intergenic is-ncRNAs, qRT-PCR analysis was also performed on a Rotor-Gene Q apparatus (QIAGEN) using the supplied software and TransScript II Green One-step qRT-PCR SuperMix (Transgen) according to the manufacturer's instructions. The primers used in these experiments are listed in DNase I-treated total RNA (300 ng) from the early and late intraerythrocytic stages of http://www.ncbi.nlm.nih.gov/geo/). The accession number for GEO is GSE45172.The data obtained from the Illumina/Solexa paired-end sequencing in this study was deposited in the Gene Expression Omnibus database at NCBI . Table S4. The 60 novel is-ncRNAs confirmed by RT-PCR. Table S5. Annotations of the cis-encoded sense RNAs of the novel antisense RNAs. Table S6. Oligos used to remove known ncRNAs. Table S7. Primers used for RT-PCR. Table S8. Oligos used to obtain RNA probes in northern blotting assays. Table S9. Primers used for qRT-PCR. Table S10. Ct values found for the qRT-PCR.(XLS)Click here for additional data file.File S2Figure S1. Internal motifs (IM1 and IM2) of the novel intergenic is-ncRNAs and similar RNA motifs in Homo sapiens. Figure S2. RT-PCR confirmation of additional 50 novel is-ncRNAs. Each is-ncRNA is represented by three adjacent lanes; from left to right, these lanes include DNase-treated RNA RT-PCR (“+”), RT-PCR with no RNA template  and RT without reverse transcriptase . “M” indicates the 50 bp DNA ladder. “Length” indicates the expected sizes of the is-ncRNAs based on the Illumina/Solexa paired-end sequencing assembly data.(PDF)Click here for additional data file.